Time-Resolved Analysis of Biological Reactions Based on Heterogeneous Assays in Liquid Plugs of Nanoliter Volume

Abstract

In this article, we present a concept which uses liquid plugs as reaction volumes for heterogeneous assay reactions to facilitate time-resolved analysis of biomolecular reactions. For this purpose, the reaction is first compartmentalized to a train of many identical plugs. Therefore, we established a simple fluidic setup build from off-the-shelf available tubing and connectors. It permits reliable formation of plugs and successive dosing of further assay reagents to these compartments (plug volume <5% CV). The time course of the reaction is obtained by routing the plugs successively through a detector. Thereby, the arrival time of a given plug at the detector represents the reaction time of the overall reaction at that moment. Thus, each analyzed plug represents a discrete state of the overall reaction. With this approach, we can achieve a temporal resolution as small as one second, which hardly can be met by conventional analytical methods for analysis of endogenous biological compounds. For analysis of the content of the plugs, we developed a method which allows for heterogeneous assays in two-phase flow. For this purpose, functionalized superparamagnetic beads are enclosed in the plugs for specific binding of the assay product. Purification from supernatant species is achieved by transferring the beads with bound analyte across the phase boundary between aqueous plugs and water-immiscible carrier fluid. We demonstrate this assay principle exemplarily for a sandwich immunoassay (cytokine IL-8). Time-resolved analysis is validated by monitoring a cell-free in vitro expression reaction (turboGFP) in plugs and conventionally in bulk solution. We show that our approach allows for analyzing the entire course of a reaction in a single run. It permits kinetic studies of biological processes with significantly reduced experimental effort and consumption of costly reagents.

Cell-Free Synthetic Biology: Engineering Beyond the Cell

“…Fluorescent RNA aptamers have also been used as an alternative simple strategy for monitoring RNA in real-time (Chizzolini et al 2014). High-throughput analysis of biomolecular interactions can also be performed by encapsulating CFPS reactions into nanoliter droplets and examining interactions via an immunoassay (Rendl et al 2013). Additionally, HPLC analyses for monitoring metabolite concentrations are straightforward to use because CFPS reactions can be run directly on an HLPC or MS machine.…”

The emerging age of cell‐free synthetic biology

“…CF-synbio expands the available tools for in situ monitoring beyond fluorescent protein expression. Examples include bioluminescence, FRET, and aptamer-based binding assays [46,47]. One elegant approach made possible by CF was recently demonstrated by Rendl and coworkers, who employed microfluidic reactors with the capability of online real-time sandwich tagging assays [47].…”

“…Examples include bioluminescence, FRET, and aptamer-based binding assays [46,47]. One elegant approach made possible by CF was recently demonstrated by Rendl and coworkers, who employed microfluidic reactors with the capability of online real-time sandwich tagging assays [47]. Similar to in vivo, in situ monitoring of CF systems relies heavily on spectrophotometry.…”

“…Recent strides in microfluidic technologies provide ultrahigh-throughput mechanisms using in vitro compartmentalization (IVC). Femtoliter microfluidic reactors, such as emulsions or plugflow modes, have been utilized for synthetic cell facsimiles [50][51][52], online aptamer assays [47], probing in vitro expression noise [53], and probing complex genetic circuits at steady state [34]. IVC reactions have enabled 10 6 genes to be analyzed with just 150 lL of reagents [54].…”

fM to aM nucleic acid amplification for molecular diagnostics in a non-stick-coated metal microfluidic bioreactor

“…Jishan13 et al used gold nanoparticle aggregates for highly sensitive identification of single-nucleotide polymorphisms in a mutated target at a concentration as low as 70 fM. Daniel et al introduced ultra-small volume analysis, ranging from micro- to nanoliters1415161718. Xueen et al demonstrated micro-loop-mediated isothermal amplification (μLAMP) with a sample volume of 0.4 μL under isothermal conditions (63°C) in 201019.…”